Method of separating cells from a sample

ABSTRACT

Rare cells are separated from a sample fluid by a positive selection or negative selection antibody by centrifuging in a tube containing a harvesting float. The harvesting float has an axial passage and a density to settle in the sample fluid and expand the layer of the target component. The positive selection antibody is preferably coupled to a particulate carrier, such as a microbead, to attach to the target component. The negative selection antibody forms a complex or conjugate with a contaminating component. In the positive separation, the particulate carrier is recovered in the axial passage of the float.

This application is a continuation of U.S. patent application Ser. No.09/756,590 filed Jan. 8, 2001, which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention is directed to a method of separating a targetcomponent and particularly target cells from a sample. Moreparticularly, the invention is directed to a method of separating targetcells from a biological sample by positive or negative separation andcentrifugation.

BACKGROUND OF THE INVENTION

Numerous methods are known in the art for separating variousconstituents from biological fluids, and particularly blood samples. Forexample, the analysis of blood components typically involves thecentrifugation of anti-coagulated whole blood to separate the cells fromplasma and to separate the various cells into layers according to thedensity of the cells. After centrifugation, the plasma fraction isremoved from the sample. Blood collection is often performed in anevacuated tube and then cell separation is achieved by centrifugation ofthe collection tube. The tube can contain a separator body that is madeof a plastic material with a specific gravity that will enable theseparator to settle during the centrifugation step onto the top of theformed component layer in the blood sample. The separator preventsmixing of the formed and unformed component fractions in the centrifugedblood sample. The separator also stabilizes the centrifuged layers forseparation and analysis.

Another method of recovering cells from a blood sample uses a hollowinsert placed in the centrifuge tube that contains the sample prior tocentrifugation. The insert is made of a transparent plastic material andfits within the centrifuge tube. The insert slides within the tube whencentrifuged to force the sample into the bore of the insert. The cellsto be harvested from the sample collect in the bore of the insertthereby forming layers of constituents that separate according to thespecific gravity of the constituents. The bore of the insert has adimension to cause the layers to elongate in comparison to the thicknessof the layer that would otherwise form in the tube without the insert.The resulting layers in the bore can be differentiated and removed fromthe bore using a hypodermic syringe or other cannula. An example of thisprocess and device are disclosed in U.S. Pat. No. 5,393,674 to Levine etal.

Another method and apparatus for separating constituents from a sampleare disclosed in U.S. Pat. No. 5,707,876 to Levine. This device uses oneor more boundary makers that are placed in the tube beforecentrifugation. The markers slide within the tube when centrifuged andidentify boundaries of the constituent layers that gravimetricallyseparate during centrifugation. A cannula is inserted into the tubethrough an elastomeric cap for injecting a liquid or gas into the tube.The injected material displaces the centrifuged sample and the boundarymarkers to one end of the tube to express the centrifuged sample fromthe tube.

Other methods of separating components from a biological sample useparamagnetic microbeads having an antigen coupled thereto. The sample ismixed with the microbeads and incubated to bind the constituent to themicrobead. The sample is then subjected to magnetic separation. Anexample of this type of method is disclosed in U.S. Pat. No. 5,916,818to Irsch et al.

These prior processes have been generally effective for their intendedpurpose. However, there is a continuing need in the industry forimproved methods for separating cells from a biological sample.

SUMMARY OF THE INVENTION

The present invention is directed to a method for separating cells froma sample, and particularly a biological sample. Accordingly, a primaryobject of the invention is to provide a method for harvesting a specificconstituent from a biological sample.

Another object of the invention is to provide a method for theseparation of a specific constituent from a biological fluid in higherconcentrations than can be obtained by prior methods.

A further object of the invention is to provide a method for harvestingselected cells from a biological fluid with low levels of contaminatingconstituents.

Still another object of the invention is to provide a method forharvesting rare cells from a biological fluid where the harvested rarecells are substantially free of mononuclear cells.

Another object of the invention is to provide a method for harvestingcells from a biological sample using a particulate carrier having acoating of an antibody having an affinity for a target cell in thesample.

A further object of the invention is to provide a method for harvestinga target constituent from a biological sample using microbeads coatedwith an antibody having an affinity for white blood cells.

Another object of the invention is to provide a method of separating atarget component from a biological sample by centrifuging the sample inthe presence of a float having an axial bore after combining the samplewith a binding agent having an affinity for at least one component ofthe sample.

Still another object of the invention is to provide a method forharvesting a target component from a biological sample by centrifugingthe sample in the presence of a particulate carrier having a positive ornegative selectivity for the target component.

Another object of the invention is to provide a method of harvestingcells from a biological sample by mixing the sample with an amount ofcarrier particles containing an antibody having an affinity for whiteblood cells and where the particles have a density greater than thedensity of white blood cells for removing white blood cells from thesample.

A further object of the invention is to provide a method of harvestingtarget cells from a biological sample by mixing the sample with anamount of carrier beads containing an antibody having an affinity forthe target cells and the beads having a density less than the density ofwhite blood cells for removing the target cells from the sample.

Still another object of the invention is to provide a method forseparating target cells from a sample and detecting the target cells ina tube, where the target cells are separated by mixing the sample withcarrier beads having an affinity for either the target cells orcontaminating cells.

The objects and advantages of the invention are basically attained byproviding a method of harvesting components from a sample material. Themethod comprises the steps of providing a sample material in a samplingcontainer, the sampling container having a focusing device with apassage for receiving and elongating layers of sample components to beharvested from the sample, providing at least one antibody in thesampling receptacle, and mixing the antibody with the sample, whereinthe antibody has an affinity for binding with at least one substance inthe sample, and centrifuging the container and sample at sufficient Gforces to separate components from the sample and to force a targetcomponent from the sample into the through passage.

The objects of the invention are further attained by providing a methodof harvesting a target component from a whole blood sample. The methodcomprises the steps of providing a whole blood sample in a samplingtube, the sampling tube containing a float dimensioned to fit within thesampling tube and having a through passage for receiving and elongatinglayers of blood constituents to be harvested from the sample, mixing thesample with at least one particulate carrier containing an antibodyhaving a binding affinity for a specific sample constituent,centrifuging the tube and sample at sufficient G forces to move thefloat toward one end of the tube and to force a target component fromthe sample into the through passage, and removing the target componentfrom the through passage.

The objects of the invention are also attained by providing a method ofharvesting a target component from a whole blood sample. The methodcomprises the steps of providing a whole blood sample in a samplingtube, the sampling tube containing a float dimensioned to fit within thesampling tube and having a through passage for receiving and elongatinglayers of blood constituents to be harvested from the sample, mixing thesample with an amount of first carrier beads having a coating of a firstantibody that has a binding affinity for a target constituent in thesample and an amount of second carrier beads having a coating of thesecond antibody that has a binding affinity for white blood cells,centrifuging the tube and sample at sufficient G forces to move thefloat toward one end of the tube and to force the first carrier beadsand target constituent into the through passage, and removing the firstcarrier beads and target constituent from the through passage.

These objects, advantages and other salient features of the inventionwill become apparent from the in view of the annexed drawings and thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings in which:

FIG. 1 is a cross-sectional side view of the centrifuge tube in oneembodiment of the invention showing a float member positioned in thetube;

FIG. 2 is a cross-sectional view of the tube of FIG. 1 aftercentrifugation;

FIG. 3 is a perspective view of a centrifuge separation device inanother embodiment of the invention;

FIG. 4 is a top view of the float of the embodiment of FIG. 3; and

FIG. 5 is a side view in cross-section of the float taken along line 5-5of FIG. 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to methods for harvesting a targetcomponent and particularly target cells from a biological sample. Moreparticularly, the invention is directed to methods for harvesting rarecells from a biological sample with fewer contaminating cells present inthe harvested rare cells.

In a preferred embodiment, the method of harvesting a target componentutilizes at least one binding agent capable of binding with a componentof the sample to assist in isolating or enhancing the target componentduring centrifugal separation. The binding agent can be an antibodyselected to have an affinity for either the target component or one ormore contaminating components. In preferred embodiments of theinvention, the affinity binding agent, such as an antibody, is providedas a coating on a particulate carrier having a particle size and densitythat is compatible with the component being harvested to enhanceseparation and recovery of the target component from the sample. In theembodiments of the invention discussed below in greater detail, theparticulate carrier can have a density lighter or heavier than thedensity of the contaminating constituent of the sample.

In further embodiments, the method is directed to harvesting rare cellsand particularly tumor cells from biological samples and particularlyanticoagulated whole blood samples. The method can be used to recover avariety of other cell types, such as stem cells and fetal cells, fromblood samples.

The method of the invention in one embodiment subjects the biologicalsample to centrifugal separation, where the sample is mixed with aparticulate carrier having an antibody or other affinity binding agentsbound to the surface of the particulate carrier. The centrifugation steppreferably uses a centrifuge harvesting device. The antibody ispreferably provided as a coating on the surface of the particulatecarrier. The particulate carrier in preferred embodiments is an amountof microbeads made of a suitable nonreactive plastic resin. Examples ofsuitable plastic resins include polystyrene, polydivinylbenzene andpolyvinylchloride.

The microbeads for use in the method of the invention can be produced byvarious methods as known in the art. In embodiments of the invention,the microbeads have a particle size ranging from about 0.05 microns toabout 7 microns, and typically about 4 microns to about 5 microns.

The particulate carrier, such as the plastic microbeads, have a densitythat complements the various components of the sample to enableenrichment of the target component and particularly to enable enrichmentof rare cells. In one embodiment, the particulate carrier includes anantibody having a binding affinity for the target component, such astumor cells. In further embodiments, other affinity binding agents canbe used. It was generally believed that after centrifugation, tumorcells are concentrated at the interface of the platelet/plasma regionand above the denser majority while cells. The tumor cells harvestedgenerally have a high concentration of contaminating mononuclear cells.It has now been found that tumor cells are not always concentrated atthe interface between the cell layers and are difficult to recoverwithout significant contamination from other interfering cells. In oneembodiment of the invention, the particulate carrier has a density thatis lower than the density of white blood cells so that the particulatecarrier and the tumor cells that are bound to or captured on the carrierare concentrated above the layer of white blood cells.

In a first embodiment of the invention, the method of harvesting andenriching a target component is a positive selection process comprisingthe steps of contacting the sample fluid with a binding agent that isable to bind with the target component, and centrifuging the sample witha device capable of expanding constituent layers and enriching thetarget component. The target component can then be harvested and furtherprocessed to identify and culture the component by known methods.Examples of suitable identifying processes include flow cytometry andmolecular nucleic acid amplification.

The centrifugation in one embodiment is carried out using a centrifugedevice 10 as shown in FIGS. 1 and 2. Device 10 in the embodimentillustrated, includes a container, such as tube 12, that is preferablymade of glass or other transparent material, such as plastic. Tube 12has a closed bottom end 14 and an open top end 16. A stopper 18 isfitted in open top end 16 to close tube 12. Preferably, stopper 18 ismade of a suitable elastomeric or rubber-like material that can bepierced by a cannula, needle or other piercing device. Stopper 18 has asubstantially cylindrical body portion 20 having an outer dimension toform a snug friction fit in top end 16 of tube 12. A shoulder 21 extendsradially outward from an upper end of stopper 18 to engage top end 16 oftube 12.

Tube 12 has a length and diameter suitable for centrifuging a samplefluid. In one embodiment, tube 12 has a length of about 75 mm, aninternal diameter of about 40 mm and a capacity of about 0.9 ml.

A float 22 is disposed in tube 12 as shown in FIG. 1. Float 22 isdimensioned to fit snugly in tube 12 and slide along the length of tube12 during centrifuging of the sample. Float 22, in the embodimentillustrated, includes an outer sleeve 24 having a cylindrical shapecomplementing the inner surface of tube 12. Outer sleeve 24 ispreferably made from a pliable material, such as a vinyl resin, that isable to deform slightly during centrifugation. Outer sleeve 24 canexpand and contract in response to the centrifugal force so that float22 is able to slide within tube 12. The pliable material returns to itsoriginal shape and dimensions at static conditions so that outer sleeve24 snugly contacts the inner surface of tube 12 and is able to slidewithin tube 12 under centrifugal force. A silicone lubricant can beapplied to the inner surface of tube 12 to assist in the slidingmovement of the float 22.

Float 22 includes an inner sleeve 26 having an axial passage or bore 28forming a through passage. Inner sleeve 26 is coupled to outer sleeve 24by a bonding agent or other suitable method. Inner sleeve 26 is made ofa rigid material that is dimensionally stable during centrifugation sosubstantially no distortion occurs during centrifugation. Axial bore 28has a length and diameter suitable for expanding a fraction of thesample material during centrifugation. In one embodiment of theinvention, axial bore 28 has an inner diameter of about 1.265 mm and alength of about 4.0 mm. Inner sleeve 26 is preferably made from aplastic such as polystyrene that does not interfere with the componentsof the sample. Float 22 is intended to be exemplary of a suitablecentrifuge device capable of separating and expanding a cell fraction.It will be understood that there are other devices that can be usedduring centrifugation to separate rare cell fractions. Suitable devicestypically include a passage or a constricted area for elongatingconstituent layers during centrifugation to enable separation of theconstituent layers.

Float 22 is dimensioned to fit in tube 12 and slide within the tube 12while centrifuging to settle between selected density layers of thesample fluid and force the target component into the axial bore 28 offloat 22. Axial bore 28 of float 22 has an internal volume suitable tocollect a substantial portion of the target component. The internalvolume and diameter of the float effectively expand the layer of thetarget component. An example of this type of cell harvesting device isdisclosed in U.S. Pat. No. 5,393,674 to Levine et al., which is herebyincorporated by reference in its entirety.

Float 22 is selected to have a density to complement the targetcomponent and the sample so that the target component collects in theaxial bore 28 by enabling float 22 to settle at a predetermined point inthe sample. In one embodiment of the invention, float 22 has a densityto settle between the resulting plasma layer and the layer of red bloodcells after centrifugation. In this embodiment, the rare cells thatnormally collect at the interface between the plasma and red blood celllayers collect in the axial bore 28 where they can be recovered.

The sample is centrifuged at a rate sufficient to separate the variousconstituents into layers. The centrifuge can be at a speed to produce acentrifugal force of about 400 G to about 800 G depending on the samplefluid. In embodiments, the centrifuge can produce a force of 1,000 G ormore.

The method of the invention can be a positive selection or a negativeselection for harvesting components, and particularly rare cells. In thepositive selection method, a sample fluid is mixed with at least oneantibody having an affinity for the target component. A negativeselection mixes the sample with an antibody having an affinity for thecontaminating cells, such as leukocytes and/or red blood cells. Suitablenegative selection antibody reagents in the form of specializedconjugates and complexes are available from StemCell Technologies Inc.and Miltenyi Botcher GmbH. Underivatized antibodies are available fromother sources such as Pharmagen, Inc. The resulting mixture is thencentrifuged using the harvesting float device to harvest the enrichedtarget component. Prior to centrifuging, the sample can be combined witha density gradient media as known in the art to enhance the separationof the target component.

The positive selection harvesting method in one embodiment of theinvention utilizes a particulate carrier having a coating of antibodywith an affinity toward the target component. In preferred embodiments,the particulate carrier is an amount of plastic microbeads with acoating of an antibody with a binding affinity for the rare cells to beharvested, and particularly tumor cells.

The density of the microbeads and the density of the float arecoordinated to collect the microbeads in the axial bore of the float.The positive selection harvesting uses microbeads having a particle sizeof about 0.05 microns to about 7 microns, and typically about 4 to 5microns, and a density less than white blood cells. The microbeads canhave a density in the range of about 1.00 to about 1.05, and preferablyabout 1.02 to about 1.03. The microbeads and the captured rare cellshave a density so that they are focused in the float when the sample andfloat are centrifuged. The float has an appropriate density so that thefloat settles in the sample after centrifuging where the rare cellsnormally settle. In this embodiment, the microbeads are sufficientlylight to float above the centrifuged layers of white and red bloodcells. The float preferably has a density to float above the white andred blood cell layers to harvest the microbeads.

The microbeads are made of a suitable material that is non-reactive withthe target component and particularly non-reactive with the rare cells.Suitable materials include polyacrylamides, polyurethanes, polysulfones,fluorinated or chlorinated resins, such as polyvinylchloride,polyethylene, polypropylene, polycarbonates and polyesters. The particleis typically about 4 to 5 microns, although the particle size can varydepending on the target component and internal diameter of the float. Anumber of commercially available microbeads have an antigen bonded tothe surface of the microbeads. The antibody can be bonded directly tothe surface of the microbead or through an intermediate coupling agent.Suitable antibody coated microbeads are commercially available fromMiltenyi Biotec GmbH. An example of a suitable microbead is availablefrom Miltenyi Biotec under the tradename MACS CD 27.

The antibody in the positive selection method has a binding affinity forthe rare cells and is selected according to the target rare cells to beharvested from the sample. Examples of rare cells to be harvestedinclude tumor cells, fetal cells and the like. Examples of tumor cellsthat can be bound to the particulate carrier can be of epithelial originand can be localized or non-localized. The tumor cells can be of thebladder, brain, breast, colon, kidney, liver, lung, ovary, pancreas,prostate, rectum and stomach. Tumor cells can also be in the form ofsarcoma, such as fibrosarcoma or rhabdosarcoma, hematopoietic tumor ofthe lymphoid or myeloid lineage, melanoma, teratocarcinoma,neuroblastoma, or glioma.

The microbeads preferably have a surface area sufficient to contain anamount of the selected antibody to bind an effective amount of the rarecells being targeted. The amount of the microbeads combined with thesample can vary with the affinity of the antibody, concentration of therare cells in the sample, the nature of the sample, and the volume ofthe sample.

The method of the invention is suitable for use in harvesting rare cellsfrom various bodily fluids, and particularly anticoagulated blood. Otherfluids that can be analyzed for rare cell content include urine, saliva,lymph fluid, spinal fluid, semen, amniotic fluid, cavity fluids andtissue extracts.

The method is carried out using the centrifuge tube 12 and float 28. Inpreferred embodiments, tube 12 is evacuated or filled with an inert gasat a subatmospheric internal pressure. The sample to be tested istransferred from a primary collecting tube by a transferring devicehaving a double piercing needle or cannula. The needle extends from thetransferring device to the tube by piercing the stopper in tube 12. Thelow pressure in tube 12 draws the fluid sample into tube 12. Athixotropic gel can be provided in tube 12 as known in the art topreserve band formation in the sample when centrifuged. Various otherseparation agents, dyes and the like can be added to tube 12 to promoteseparation and identification of components. The microbeads containingthe antibody are provided in the tube 12 and are mixed with the fluidsample by gentle shaking or stirring. The sample is then incubated tobind the target component to the microbeads.

The tube, float and sample are centrifuged at a sufficient speed and fora length of time necessary to separate the constituents of the sampleinto layers and force the microbeads and the trapped target componentinto the axial bore of the float. The sample can be centrifuged at aspeed to provide sufficient centrifugation force to cause separation ofthe layers. The tube is slowly stopped and removed from the centrifuge.A needle or cannula then pierces the stopper and is inserted into theaxial bore to remove the sample containing the microbeads. The harvestedsample is further processed and analyzed by various processes as knownin the art. In one embodiment, the harvested cells are analyzed using aflow cytometer. The rare cells or other target components can be washedand separated from the microbeads and the binding antibody by knownmethods. The resulting harvested rare cells are significantly enrichedcompared to many prior processes and have a substantially lowercontaminant level of red and white blood cells.

In a second embodiment of the invention, the method is a negativeselection method for the enrichment of rare cells. The rare cells areenriched using a binding agent that is able to bind with thecontaminating non-rare cells, such as red blood cells or white bloodcells.

In preferred embodiments, the binding agent is able to bind to one ormore white blood cell and/or red blood cell or that bind to surfaceantigens on the cells. The binding agents can be antibodies that areable to agglutinate the white blood cells or bind the white blood cellsto red blood cells. The resulting larger and denser particles can beseparated from the non-rare cells during centrifugation. Suitableantibodies that are able to bind with and capture the non-rare cellsinclude antihuman antibodies. Examples of suitable antibodies that canbe used to bind with white blood cells (leukocytes) include theleukocyte CD antibodies such as CD2, CD3, CD4, CD5, CD7, CD8, CD11a,CD11b, CD11c, CD14, CD15, CD16, CD19, CD20, CD28, CD36, CD42a, CD43,CD44, CD45, CD45R, CD45RA, CD45RB, CD45RO, CD57 and CD61. Other bindingagents that can be used include a mixture of antihuman CD45, antihumanCD19, antihuman CD14 and antihuman CD3.

Preferably, the antibodies are bound to the surface of the microbeads asin the previous embodiments. The microbeads in the negative selectionprocess have a particle size suitable for the sample and the targetcomponent. Generally, the particle size ranges from about 0.05 micronsto about 7 microns, and preferably about 4 microns to about 5 microns.In this embodiment, the beads preferably have a density greater than thedensity of white blood cells, and more preferably of about 1.07 to about1.09 g/ml, and typically in the range of about 1.08 to about 1.09 g/ml.In this manner, the microbeads sink during centrifugation and the rarecells settle above the red and white blood cell layers. The float hasdensity to float on the non-rare cells layers so that the rare cellssettle in the axial bore of the float where they can be removed.

The method of the negative selection harvesting is similar to thepositive selection discussed above. The sample fluid is provided in thetube and mixed with the microbeads containing the non-rare cellantibodies. After incubating, the tube containing the mixture isincubated and centrifuged for sufficient time to cause the layers toseparate and the rare cells to collect in the axial bore of the floatwhere the rare cells can be recovered.

In further embodiments of the invention, the method employs twomicrobeads having different affinity binding agents for capturing twodifferent components. In one embodiment, an amount of first microbeadshaving an affinity binding agent with an affinity for rare cells, suchas tumor cells, are mixed with the sample. The first microbeads have adensity to separate from the white and red blood cells. The firstmicrobeads have a particle size, density and affinity binding agentsubstantially the same as the microbeads of the positive selection ofthe previous embodiment. An amount of second microbeads having anaffinity binding agent with an affinity for white blood cells is alsomixed with the sample. The second microbeads have a density to separatethe white blood cells, red blood cells, or a combination thereof fromthe rare cells. The resulting mixture is centrifuged with the float sothat the first microbeads with the captured rare cells settle in theaxial passage where they can be recovered. The second microbeads have aparticle size, density and affinity binding agent substantially the sameas the negative selection method of the previous embodiment. In thismanner, the second microbeads separate from the first microbeads duringcentrifugation to separate the contaminating cells, such as the redand/or white blood cells from the target cells and the first microbeads.The first microbeads have a particle size and density to be collected inthe axial passage of the float for recovering the target cells.

Referring to FIG. 3, another embodiment of the centrifuge device 30 isshown. Device 30 is particularly suitable for various cell manipulationsafter separation from a sample. For example, rare cells can be separatedfrom a sample and subjected to various detection and assay processeswith device 30. In this embodiment, device 30 includes a hollowcontainer 32 having a substantially rectangular shape. Container 32includes a front wall 34, and an opposite rear wall 36 having alongitudinal length and a width. Opposite side walls 38 and a bottomwall 40 extend between front wall 34 and rear wall 36 to form an opencavity 42. Container 32 includes an open end 44 to receive a stopper 46for closing cavity 42. Preferably, container 32 is made of a transparentmaterial such as glass or plastic.

Container 32 is dimensioned to receive a volume of a biological samplesuitable for analysis of a target component. Container 32 generally hasa volume of about 8 ml to about 10 ml, and preferably about 9 ml. In theillustrated embodiment, side walls 38 of container 32 have a dimensionto define a thickness of cavity 42 that is sufficiently thin tovisualize, detect and analyze a target component through front wall 34.Examples of suitable detection and analysis methods include microscopyto visualize cells in the sample. Container 32 is typically about 7 cmto about 8 cm in length, and about 3 cm to about 4 cm in width. Sidewalls 38 are dimensioned so that cavity 42 has a thickness of about 3 mmto about 6 mm, and preferably about 4 mm.

Container 32 includes a movable float 48 that is able to slide withincontainer 32 in the longitudinal dimension in a manner similar to theprevious embodiment. Float 48 is dimensioned to fit within cavity 42 ofcontainer 32 and has an outer dimension corresponding to the innerdimension of container 32. As shown in FIGS. 3-5, float 48 has a base 50with a substantially flat bottom surface 54. Base 50 includes aninclined leading end 56 and an inclined trailing end 58. A plurality ofribs 60 are coupled to top surface 54 of base 50.

As shown in FIG. 5, ribs 60 extend in a longitudinal direction withrespect to the longitudinal dimension of base 50. Ribs 60 are aligned inpairs to form channels 62 extending the length of base 50 betweenadjacent ribs. Ribs 60 have a height to fit closely against the innersurface of container 32. Channels 62 are dimensioned to separate andelongate the layers during centrifugation.

In this embodiment, a biological sample, such as a blood sample, isplaced in container 32. An amount of microbeads 64 having an affinitybinding agent for a target component is mixed with the sample. Container32 is then centrifuged as in the previous embodiment to collect themicrobeads 64 with the captured target component in longitudinalchannels 62 of float 48. Microbeads 52 and the captured target componentcan then be analyzed by visualizing the target component withincontainer 32 by microscopy methods as known in the art.

In the embodiments shown in FIG. 5, the blood sample after centrifugingseparates into a layer of red blood cells 66, a granulocyte cellfraction layer 68, a mononuclear cell fraction 70, a plasma fraction 72and a platelet/plasma interface 74. In a positive selection process,microbeads 64 have an affinity for the target compound and collect inchannels 62. Alternatively, microbeads 64 can have an affinity for whiteand/or red blood cells in a negative selection process. Channels 62 areformed between ribs 60 and are enclosed by top wall 34 of container 32.Inclined leading edge 56 and inclined trailing edge 58 divert the samplethrough channels 62 as float 48 slides through container 32. Microbeads64 are retained in a thin layer in channels 62 close to top wall 34 ofcontainer 32 so that the microbeads 64 can be visualized through topwall 34 by microscopy or other analytical methods as known in the art.Preferably, front wall 34 of container 32 is substantially flat toprevent the optical distortion normally associated with cylindricalcontainers.

EXAMPLE

This example compares the harvested tumor cells from a sample with andwithout a negative selection. The harvester separations were comparedfor 6.0 mls of freshly collected whole blood that were spiked with 0, 50and 500 cultured prostate tumor PC-3 cells. An antibody cocktailobtained from StemCell Technologies, Inc. under the tradename RosetteSepwas mixed with each of the blood samples and incubated for 20 minutes atroom temperature. A control blood sample was prepared without theantibody treatment. The antibody cocktail provided a negative selectionto remove the unwanted white blood cells.

The blood samples were layered on top of a density media in a 16×100 PETVacutainer tube obtained from Becton Dickinson containing a harvesterfloat and 2 mls of POLYMORPHPREPTM density media. The samples werecentrifuged in a swinging bucket centrifuge for 30 minutes at 20□C at arate of about 650 g. The control sample showed the presence of whiteblood cells with the tumor cells in the harvester float. The antibodytreatment demonstrated tumor cells collected in the harvester float witha greatly reduced white cell population. The tumor cells were removedfrom the harvester float. Flow cytometry demonstrated recovery of about90% of the tumor cells.

While various embodiments have been chosen to illustrate the invention,it will be appreciated by those skilled in the art that variousmodifications and additions can be made without departing from the scopeof the invention as defined in the appended claims.

1. A method of separating at least one target component from abiological sample, said method comprising a. placing said biologicalsample into a separation container, said separation container comprisinga focusing device, having an axial bore passage and being capable ofvertical movement within said separation container upon centrifugationand a specific density a set of selection microbeads having at least onetarget affinity binding agent bound to their surfaces, said at least onetarget affinity binding agent having a binding affinity for said atleast one target component within said biological sample and a negativeselection binding agent having a binding affinity for a component otherthan said target component within said biological sample, such that acomplex or conjugate is formed between said negative selection bindingagent and said component other than said target component within saidbiological sample on contact with said biological sample, wherein thespecific density of said focusing device is substantially equal to thedensity of said set of selection microbeads and the specific density ofsaid complex or conjugate formed between said negative selection bindingagent and said component other than said target component within saidbiological sample is different to that of said focusing device and saidset of selection microbeads b. centrifuging said separation containercontaining said biological sample to densitometrically separatecomponents of said sample into layers such that separation of said setof selection microbeads and said complex or conjugate formed betweensaid negative selection binding agent and said component other than saidtarget component within said biological sample is induced, wherein atarget layer comprising said set of selection microbeads bound to saidat least one target component is located within said axial bore passageof said focusing device and wherein said complex or conjugate formedbetween said negative selection binding agent and said component otherthan said target component within said biological sample is absent fromsaid axial bore passage of said focusing device after centrifugation;and c. aspirating said elongated target layer to remove said at leastone target component from said separation container.
 2. The method ofclaim 2, further comprising mixing said biological sample with said setof selection microbeads and said negative selection binding agent priorto centrifugation.
 3. The method of claim 2, wherein said separationcontainer is a cylindrical, closed-end tube with an inner surface, andsaid focusing device having an outer surface that complements said innersurface of said tube.
 4. The method of claim 3, wherein said biologicalsample is blood.
 5. The method of claim 4, wherein said focusing devicecomprises consisting of a single bore axial passage.
 6. The method ofclaim 5, wherein said selection microbeads have a density of betweenabout 1.00 g/cc and about 1.06 g/cc.
 7. The method of claim 6, whereinsaid set of selection microbeads and said negative selection bindingagent each comprise at least one antibody.
 8. The method of claim 7,wherein said negative selection binding agent binds to the surface ofnormal white blood cells.
 9. The method of claim 7, wherein saidnegative selection binding agent agglutinates white blood cells.
 10. Themethod of claim 7, wherein said negative selection binding binds whiteblood cells to red blood cells.
 11. The method of claim 7, wherein saidnegative selection binding agent comprises at least one leukocyte CDantibody.
 12. The method of claim 7, wherein said antibody of said setof selection microbeads binds to the surface of cells other than saidnormal white blood cells.
 13. The method of claim 12, wherein said cellsother than normal white blood cells are selected from the groupconsisting of cancer cells and fetal cells.